WO2014141111A1 - Methods for prevention and treatment of graft-versus-host disease - Google Patents

Abstract

Disclosed are compositions and methods for treating graft-versus-host disease.

Description

METHODS FOR PREVENTION AND TREATMENT OF GRAFT- VERSUS-HOST

DISEASE

This Application claims the benefit of U.S. Provisional Application No. 61/781,205, filed on March 14, 2013 and U.S. Provisional Application No. 61/821,253, filed on May 9, 2013, each of which is incorporated herein by reference in their entirety.

I. BACKGROUND

1. Graft-versus-host disease (GVHD) is a common complication following an allogeneic tissue transplant and occurs in approximately 50% of transplant recipients. Acute GVHD is a major source of morbidity and mortality following allogeneic hematopoietic cell transplantation.

2. Approximately 25,000 allogeneic hematopoietic cell transplants (bone marrow, peripheral blood stem cell [PBSC], or cord blood transplants) are performed annually worldwide. Over time, a continuous increase has occurred in the number of unrelated donors and in the number of allogeneic transplants for AML, ALL, MDS, and lymphomas. There is also an increase in the allogeneic transplantations for non-malignant diseases and in patients over 50 years of age.

3. The global incidence of acute GVHD ranges from 26% - 34% in recipients of fully matched, sibling donor grafts to 42% - 52% in recipients of matched, unrelated donor grafts. Evidence from the US suggests that incidence ranges from 30% in recipients of fully

histocompatible transplants to 60% - 70% in recipients of mismatched hematopoietic cells or hematopoietic cells from an unrelated donor.

4. There is no FDA approved treatment for either acute or chronic GVHD. Treatment strategies for acute GVHD aim to reduce the immune reaction to the donor T cells and therefore includes immunosuppressive treatment like cyclosporine, high dose steroids, and methotrexate. The standard therapy for de novo acute GVHD is high dose methylprednisolone, with expected response rates of 18% - 50%. For patients who develop steroid-refractory acute GVHD, there is no standard of care therapy, and expected survival is less than 30%. Therefore, novel therapies are urgently needed for this patient population.

II. SUMMARY

5. Disclosed are methods and compositions related to methods and compositions for use in treating graft versus host disease. In one embodiment, disclosed herein are methods of treating, inhibiting, or preventing graft-versus-host disease (GVHD) in a subject in need thereof, the method comprising administering to said subject a therapeutically or prophylactically effective amount of adherent stromal cells, thereby treating or preventing the GVHD.

6. Also disclosed herein are uses of adherent stromal cells for the manufacture of a medicament for treating or preventing GVHD in a subject, wherein the cells are optionally obtained from placental or adipose tissue.

7. In another embodiment, disclosed herein are adherent stromal cells for the treatment or prevention of GVHD, wherein the cells are optionally obtained from 3D culturing. In another embodiment disclosed herein are adherent stromal cells or compositions comprising adherent stromal cells, wherein the cells are derived from placental or adipose tissue.

8. Embodiments of the disclosure further relate to articles of manufacture including a packaging material including a label for use in treating or preventing GVHD, wherein the packaging material packages a pharmaceutically effective amount of adherent stromal cells from a placenta or adipose tissue. In some embodiments, the adherent stromal cells are adherent stromal cells from placenta or adipose tissue, described in International Patent Publication Numbers WO 2007/108003 and WO 2009/037690 (3D-adherent cells by Plurix; PLX cells), each of which is incorporated herein by reference herein in its entirety.

9. In some embodiments, the subject has acute GVHD. The subject may have grade I acute GVHD under the Glucksberg GVHD grading system. In some embodiments, the subject with grade I acute GVHD is receiving one or more of a topical therapy for skin rash and a calcineurin inhibitor. In further embodiments, the subject has grade II- IV acute GVHD under the Glucksberg grading system. Subjects with grade II-IV acute GVHD may be receiving a corticosteroid.

10. In some embodiments, the subject has chronic GVHD. The subject with chronic GVHD may be receiving an immunosuppressive treatment. The immunosuppressive treatment may be one or more of methotrexate, cyclosporine, a corticosteroid, and antithymocyte globulin. The corticosteroid may be methylprednisolone. In some embodiments, the subject requires or has required immunosuppressive treatment for a period of one or more years.

11. In further embodiments, the subject has steroid-refractory GVHD. The subject may be receiving one or more of extracorporeal photophoresis, anti-TNF alpha antibody, mammalian target of rapamycin (mTOR) inhibitor, mycophenolate mofetil, interleukin-2 receptor antibody, alemtuzumab pentostatin, mesenchymal stem cells, and methotrexate.

12. In some embodiments, adherent cells are administered at multiple time points and/or at variable intervals. In some embodiments, the cells are administered in a single administration. In further embodiments, the cells are administered in multiple administrations. Cells may be administered with a dosage of from about 150 million to about 300 million cells. In some embodiments, subsequent doses are lower in cell number. The schedule of administration and/or dosing may be determined in consultation with an oncologist.

13. In some embodiments, adherent stromal cells (including, for example, PLX cells) are administered to a subject systemically. In further embodiments, the cells are administered locally. The adherent cells may be administered locally at the site of a tissue transplant. In some embodiments, adherent cells are administered intramuscularly. Intramuscular (IM) administration also allows secretion of cytokines at systemically detectable levels, and offers ease and speed of delivery. In some embodiments, adherent stromal cells (including, for example, PLX cells) are administered by one or more further modes of administration, including, for example, intravenous, intraperitoneal, subcutaneous, intradermal, intraosseous infusion, and inhalation administration, and the like.

14. In some embodiments, the subject receiving adherent stromal cells (including, for example, PLX cells) has received or will receive a hematopoietic stem cell transplant. In some embodiments, the subject has a hematopoietic neoplastic disorder. The subject may have leukemia. The leukemia may be chronic myelogeneous leukemia or chronic lymphocytic leukemia. In some embodiments, the subject has lymphoma. The lymphoma may be Hodgkin's disease or non-Hodgkin's lymphoma. In some cases, the subject has multiple myeloma. The subject may receive or will receive a bone marrow, peripheral blood stem cell, or cord blood transplant. In some embodiments, the subject has received or will receive whole body irradiation. In further embodiments, the subject is female.

15. In some embodiments, the subject has received or will receive a transplant from an HLA-matched related donor or an HLA-matched unrelated donor. The subject may also have received or may receive a transplant from an HLA-mismatched related or unrelated donor.

16. The adherent stromal cells (including, for example, PLX cells) may be obtained from a three-dimensional (3D) culture. The three-dimensional (3D) culture may comprise a 3D bioreactor. In some embodiments, the adherent cells in the 3D culture are effected under perfusion. Culturing of the adherent cells may be effected for at least 3 days. Culturing may be effected until at least 10 % of the adherent cells are proliferating. In some embodiments, adherent cells comprise cells cultured from the placenta or adipose tissue under 2 dimensional (2D) culturing conditions. 17. In some embodiments, adherent stromal cells (including, for example, PLX cells) are autologous to the subject. In further embodiments, adherent stromal cells (including, for example, PLX cells) are allogeneic to the subject.

III. BRIEF DESCRIPTION OF THE DRAWINGS

18. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

19. Figure 1 shows the percent body weight following irradiation and induction of GVHD for mice receiving PLX treatment via i.v. (triangle) and i.m (square) injection and controls (diamonds).

20. Figure 2 shows the percent survival and chimerism of mice following induction of GVHD and treatment. Figure 2A shows the survival of mice receiving PLX cells via i.m.

injection (star) or i.v. injection (triangle) and negative controls (diamonds) 45 days after GVHD induction. Figure 2B shows the percent chimerism of mice in negative control group and mice receiving PLX cells via i.m. and i.v. injection.

21. Figure 3 shows the GVHD score following transplantation. Figure 3 A shows the average GVHD score over a 35 day period for negative controls (diamonds) and mice receiving PLX cells via i.m. (squares) or i.v. (triangle) injection. Figure 3B shows the median GVHD score for all three groups at Day 25 and Day 36 following transplantation.

22. Figure 4 shows the skin GVHD score following transplantation. Figure 4A shows the average skin GVHD score over a 32 day period for negative controls (diamonds) and mice receiving PLX cells via i.m. (squares) or i.v. (triangle) injection. Figure 4B shows the median skin GVHD score for all three groups at Day 32 following transplantation.

23. Figure 5 shows the GVHD score in surviving mice following transplantation. Figure 5A shows the average GVHD score in surviving mice at day 45 following transplantation for negative controls (diamonds) and mice receiving PLX cells via i.m. (squares) or i.v. (triangle) injection. Figure 5B shows the median GVHD score for surviving mice in all three groups at Day 36 and Day 43 following transplantation.

24. Figure 6 shows the fur loss associated with skin GVHD that occurred in several control mice and an IM PLX-treated mouse but not in normal mice or IV PLX-treated mice.

IV. DETAILED DESCRIPTION

25. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definitions

26. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

27. Ranges can be expressed herein as from "about" one particular value, and/or to

"about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

28. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

29. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. B. Methods of treating Graft versus Host Disease (GVHD)

30. In one aspect disclosed herein are methods of treating, inhibiting, or preventing GVHD in a subject in need thereof. Graft-versus-host disease (GVHD) is an immunological disorder in which the immune cells of a transplant attack the cells of a transplant recipient and lead to organ dysfunction. For example, it is associated with allogeneic bone marrow (BM) transplantation in which T-cells from the transplanted BM recognize the host (the bone marrow- transplanted patient i.e., the recipient) as non-self and attack its tissues and organs. The patient's own immune system is suppressed prior to the transplant to prevent rejection of the graft and therefore cannot respond to the attack. The organs most commonly attacked are the

gastrointestinal (GI) tract, skin, liver, and lungs.

31. There are three prerequisites to the development of GVHD. First, the graft must contain immunologically competent cells. Second, the host must be immunologically distinct (i.e., histo-incompatible) from the graft, such that host antigens appear foreign to the immune cells of the graft and can therefore activate these cells. Third, the host itself must be incapable of mounting an effective immune response to the graft.

32. Thus, GVHD commonly develops after an allogeneic bone marrow transplant (BMT). But it can also appear after solid organ transplantation. The exact incidence rate of GVHD after solid organ transplantation is unknown. Mild cases likely remain undiagnosed because the clinical features of fever, rash, and diarrhea can be misinterpreted as related to post- transplantation infections. The incidence rate of GVHD is highest after small bowel

transplantation (about 5%), followed by liver transplantation. But in general, the incidence rate for solid organ transplantation is very small relative to bone marrow transplantation.

33. Because GVHD is a disease where the donor's immune system attacks the recipient's body, there is a need for new methods of inhibiting the immunological assault from the donor graft. Accordingly, aspects of the disclosure further relate to a multifold mode of action for GVHD treatment, inhibition, or prophylaxis using adherent stromal cells, such as, for example, adherent stromal cells from placenta or adipose tissue, including, for example, PLX cells. Thus, in one aspect disclosed herein are methods of treating, inhibiting, or preventing GVHD in a subject in need thereof, the method comprising administering to said subject a pharmaceutically effective amount of adherent stromal cells, thereby treating or preventing the GVHD.

34. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

35. "Treatment," "treat," or "treating" mean a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from native levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition. Therefore, in the disclosed methods, treatment" can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or the disease progression. For example, a disclosed method for reducing the effects of GVHD is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject with GVHD when compared to native levels in the same subject or control subjects. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. It is understood and herein contemplated that "treatment" does not necessarily refer to a cure of the disease or condition, but an improvement in the outlook of a disease or condition (e.g.., GVHD).

Nevertheless, it is fully contemplated herein that "treatment" can not only refer to the ablation of the disease state, but the reversal of the condition. It is also understood that by correcting or improving symptoms associated with GVHD, the disease state of GVHD is being treated.

36. As used herein "preventing" or "prevention" refers to any methodology where the disease state does not occur due to the actions of the methodology (such as, for example, administration of adherent stromal cells). In one aspect, it is understood that prevention can also mean that the disease is not established to the extent that occurs in untreated controls. For example, there can be a 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100% reduction in the establishment of disease.

37. Adherent stromal cells ("ASC") are cells obtained from a tissue, including but not limited to placenta and adipose tissue, that are adherent when cultured in vitro, typically express (i.e., have positive surface marker expression ) one or more, two or more, three or more, or all four of CD105, CD73, CD90 and CD29 and lack detectable expression (i.e., have negative surface marker expression) of at least one, two , three, four, five, six, seven, eight, nine, or all ten of CD3, CD4, CD45, CD80, HLA-DR, CD1 lb, CD14, CD19, CD34 and CD79 by flow cytometry compared to an isotype control.

38. The term "placenta" refers to any portion of the mammalian female organ which lines the uterine wall and during pregnancy envelopes the fetus, to which it is attached by the umbilical cord. Following birth, the placenta is expelled (and is referred to as a post-partum placenta). In some embodiments, "placenta" refers to whole placenta.

39. In those aspects and embodiments involving placental-derived adherent stromal cells, the placental-derived ASCs may be obtained from both fetal (i.e., amnion or inner parts of the placenta) and maternal (i.e., decidua basalis, and decidua parietalis) parts of the placenta unless the context otherwise makes clear that only fetal or maternal parts are meant.

40. It is contemplated herein that it can be advantageous to administer more than a single dose of the adherent stromal cells (including, for example, PLX cells), compositions comprising said adherent stromal cells (including, for example, PLX cells), or medicaments manufactured using said adherent stromal cells (including, for example, PLX cells) in performing the methods of treatment, inhibition, or prevention of GVHD disclosed herein. In one aspect, the disclosed adherent stromal cells, medicaments, and/or compositions can be administered singly or in multiple administrations. Thus, disclosed herein are methods of treating, inhibiting, or preventing GVHD and uses comprising administering the disclosed adherent stromal cells (including, for example, PLX cells), medicaments, and/or compositions comprising said cells, wherein the adherent stromal cells, compositions comprising said adherent stromal cells, or medicament manufactured using said adherent stromal cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 24, 25, 30, 35, 36, 40, 45, 48, 50, 55, 60 or more times. When more than a single administration, the adherent stromal cells or compositions comprising said cells may be administered at regular or variable intervals. For example, the adherent stromal cells (including, for example, PLX cells) or compositions comprising said cells may be administered regularly such that they or administered hourly, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours; daily, every other day; every third day, weekly, every two weeks, monthly, or yearly.

Alternatively, cells or compositions comprising said cells can be administered according to the methods at irregular (i.e., variable intervals) such as, for example, a first dose, a second dose a week later, and a third dose a month following the first dose later; or a first dose followed by a second dose a month later and a third dose three months following the first dose; or doses at one week intervals for three months, followed by monthly intervals until the one year point, followed by yearly doses. Also disclosed are methods wherein the adherent stromal cells, compositions comprising adherent stromal cells, and/or medicaments manufactured using said adherent stromal cells concluding, for example, PLX cells) an be administered for a duration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, or 5 years.

41. Additionally, it is contemplated herein that the in the disclosed methods of treating, inhibiting, or preventing GVHD, the disclosed adherent stromal cells (including, for example, PLX cells), medicaments, and/or compositions comprising adherent stromal cells can be administered before (prophylactically), after (therapeutically), concurrently with

(prophylactically), or in combination with a graft (prophylactically). For example, the disclosed adherent stromal cells, compositions comprising said adherent stromal cells, or medicament manufactured using said adherent stromal cells can be administered 7, 6, 5, 4, 3, 2, days, 24, 23, 22, 21, 20, 19, , 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours, 45, 30, 15, 10, 5, 4, 3, 2, or 1 min or any combination thereof, before administration of the graft in the disclosed methods and uses of treating GVHD. Alternatively, the disclosed adherent stromal cells, compositions comprising said adherent stromal cells, or medicament manufactured using said adherent stromal cells can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60min, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 20, 24, 25, 30, 36, 40, 45, 48, 50, 55, or 60 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, or 60 months or any combination thereof after administration of the graft in the disclosed methods and uses of treating GVHD. It is also disclosed that the adherent stromal cells (including, for example, PLX cells), compositions comprising said adherent stromal cells, or medicament manufactured using said adherent stromal cells can be administered after onset of GVHD. Accordingly, disclosed herein are methods of treating GVHD wherein the adherent stromal cells, or medicament manufactured using said adherent stromal cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60min, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 20, 24, 25, 30, 36, 40, 45, 48, 50, 55, or 60 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, or 60 months or any combination thereof after onset of GVHD. It is further understood that adherent stromal cells (including, for example, PLX cells), compositions comprising said adherent stromal cells, or medicament manufactured using said adherent stromal cells can be administered in combination before, concurrently with, and/or after administration of the graft in the disclosed methods and uses of treating GVHD.

42. It is understood and herein contemplated that additional agents may need to be administered along with the adherent stromal cells to facilitate engraftment or further guard against GVHD. Accordingly, disclosed herein are methods for treating, inhibiting, or preventing GVHD in a subject in need thereof comprising administering to the subject adherent stromal cells, medicaments manufactured using said adherent stromal cells, and/or composition comprising adherent stromal cells, wherein the method further comprises an immunosuppressive agent, steroid, anti-inflammatory, small molecule, pharmaceutical, antibody, or other agent. For example, a topical therapy for skin rash; a calcineurin inhibitor; a corticosteroid; immunosuppressive treatment (e.g., methotrexate, cyclosporine, a corticosteroid, and/or anti- thymocyte globulin); anti-TNF alpha antibody; mammalian target of rapamycin (mTOR) inhibitor; mycophenolate mofetil, interleukin-2 receptor antibody; alemtuzumab pentostatin; and/or mesenchymal stem cells can be administered in combination with the adherent stromal cells, medicaments manufactured using said adherent stromal cells, and/or compositions comprising said adherent stromal cells. Such immunosuppressive agent, steroid, antiinflammatory, small molecule, pharmaceutical, antibody, or other agents can be administered in before, after, concurrently with or in the same combination with adherent stromal cells.

43. In one embodiment, the decision of additional agents to be administered in addition to the adherent stromal cells (including, for example, PLX cells), medicament, or compositions in the disclosed methods is determined based on whether the subject has acute or chronic GVHD and/or the grade of GVHD. Accordingly, disclosed herein are methods of treating, inhibiting, or preventing GVHD in a subject comprising administering to the subject adherent stromal cells, compositions comprising said adherent stromal cells, or medicaments manufactured using said stromal cells, wherein the subject has acute GVHD. In another embodiment, disclosed herein are methods of treating, inhibiting, or preventing GVHD in a subject comprising administering to the subject adherent stromal cells, compositions comprising said adherent stromal cells, or medicaments manufactured using said stromal cells, wherein the subject has chronic GVHD.

44. GVHD can be mild, moderate, or severe, depending on the extent of damage inflicted to different organs.

45. The disease is divided into acute and chronic GVHD according to clinical manifestations. Patients with acute GVHD typically suffer damage to the skin, GI tract, and liver. Skin damage ranges from redness to exfoliation. Insult to the GI tract can result in bloody diarrhea and blood loss. Liver manifestations, though usually cholestatic in nature, can include liver failure in rare cases.

46. Acute GVHD usually develops within the first 100 days after transplantation, but it can also occur later. The clinical manifestations of chronic GVHD include red and itchy skin, dry eyes, dry mouth, abnormal liver function with jaundice, and lung damage due to

bronchiolitis obliterans. Chronic GVHD is the major cause of non-relapse mortality after allogeneic hematopoietic transplantations. Chronic GVHD usually develops more than 100 days after transplantation, but it can appear sooner.

47. Patients with chronic GVHD require prolonged immunosupressive treatment, averaging two to three years in length. The mechanisms underlying chronic GVHD are considered to be somewhat distinct from those of acute GVHD. Thus, chronic GVHD is not simply an end-stage of acute GVHD.

Clinical Staging of Acute GVHD (aGVHD)

48. There are two systems for quantifying the severity of aGVHD, namely, the International Bone Marrow Transplant Registry (IBMTR) grading system and the Glucksberg grading system. For both systems, the stage of aGVHD is first determined separately in the three main target organs (skin, liver and gut). These grades are then used to determine an overall aGVHD grade, using either the International Bone Marrow Transplant Registry (IBMTR) or Glucksberg criteria.

49. For each grading system, the acute GVHD stage for each target organ is first determined according to certain clinical measures, as provided below in Table I.

Table I: Criteria for stages of acute GVHD

STACK

ORGAN

Skin <25% 25 - 50% >50% or Generalized

(maculopapular rash generalised erythroderma with extent, % of body erythroderma bullous formation surface area) or desquamation

Liver 34 - 50 51 - 102 103 - 255 > 255

(bilirubin mmol L) [2 - 3] [3 - 6] [6 - 15] [> 15]

[bilirubin mg/dL] ø) (or AST 150- 750 U/L)

Gut >30mL kg or >60mL/kg or >90mL/kg or >2000mL/day or

(daily diarrhea >500mL (2) >1000mL >1500mL severe abdominal volume) pain with or

without ileus

Notes:

(1) Stage is downgraded by one stage if an additional cause of hyperbilirubinemia is documented.

(2) Persistent nausea with histologic evidence of stomach or duodenal acute GVHD is assessed as stage- 1 for the gut.

50. The overall acute GVHD grade is then determined using either the IBMTR or Glucksgerg systems, as shown in tables II and II below.

D Stage 4 in any organ 12.3

Table III: Glucksberg Grading

OVERALL 1 YEA R MOR IV iLITY (ODDS

GLUCKSBERG CRITERIA RATIO COMPARED TO NO

GRADE GVH D [4])

Skin stage 1 or 2 only (no liver

acute GVHD)

Up to stage 1 liver or gut, up to stage 3

II 0.8

skin

Up to stage 4 liver, up to stage 3 in any

III 2.2

other organ

Stage 4 skin or gut, ECOG/WHO

IV performance status of 4 or Karnofsky 13.1

performance score <30%

51. The IBMTR and Glucksberg criteria were prospectively validated in 607 patients receiving myeloablative T cell-replete allogeneic stem cell transplants between 1996 and 1999. Both systems were predictive of mortality when grading was performed at 100 days and one year following transplantationA

52. A study published in 2011 by a group from Fred Hutchinson Cancer Research Center evaluated and compared risk factors for grades II- IV acute GVHD and for chronic GVHD in 2941 recipients of first allogeneic hematopoietic cell transplantation. Of the 2941 recipients, 1927 (66%) received bone marrow, 1284 (44%) had HLA-matched related donors, 957 (33%) had HLA-matched unrelated donors, and 700 (24%) had HLA-mismatched related or unrelated donors. The study reported a cumulative incidence of grades II- IV acute GVHD at 6 months of 80%. The risk of developing GVHD in allogeneic bone marrow transplant recipients is increased in the following populations: (1) patients receiving transplants of mismatched bone marrow; (while 40% of patients administered a matched transplant will suffer from GVHD, the incidence increases to 60% for patients receiving unmatched transplants); (2) patients receiving total body irradiation in the conditioning protocol of BMT; and (3) female patients. In one aspect, disclosed herein are methods of treating, inhibiting, or preventing GVHD, adherent stromal cells for use in manufacturing a medicament for treating, inhibiting, or preventing GVHD, or adherent stromal cells or compositions comprising said adherent stromal cells wherein the subject with GVHD or receiving the medicament, adherent stromal cells, or compositions comprising adherent cells has been exposed, is being exposed, or will be exposed to whole body irradiation. In another aspect, disclosed herein are methods of treating, inhibiting, or preventing GVHD, adherent stromal cells for use in manufacturing a medicament for treating, inhibiting, or preventing GVHD, or adherent stromal cells or compositions comprising said adherent stromal cells wherein the subject with GVHD or receiving the medicament, adherent stromal cells, or compositions comprising adherent cells is female, disclosed herein are methods of treating, inhibiting, or preventing GVHD, adherent stromal cells for use in manufacturing a medicament for treating, inhibiting, or preventing GVHD, or adherent stromal cells or compositions comprising said adherent stromal cells (including, for example, PLX cells) wherein the subject with GVHD or receiving the medicament, adherent stromal cells, or compositions comprising adherent cells will receive, is concurrently receiving, or will receive a hematopoietic stem cell, bone marrow, peripheral stem cell, or cord blood transplant.

53. The risk of GVHD can be lowered by reducing the intensity of the conditioning regimen, by excluding T cells from graft (which reduces graft- versus-leukemia [GVL] and increase the recurrence of malignancy), or by administering immunosuppressive treatment such as methotrexate, cyclosporine, corticosteroids, or anti-thymocyte globulin.

54. The joint working group established by the Haemato-oncology subgroup of the British Committee for Standards in Haematology (BCSH) and the British Society for Bone Marrow Transplantation (BSBMT) reviewed the available literature and made recommendations in 2012 for the management of acute graft-versus-host disease. Their recommendations are as follows: (l)The management of grade I disease should include topical therapy and optimizing levels of calcineurin inhibitors without the need for additional systemic immunosuppression. (2) The use of systemic corticosteroids is recommended for first line therapy for grade II-IV GVHD. (3)The following agents are suggested for use in the second line treatment of steroid- refractory acute GVHD: extracorporeal photopheresis, anti-tumour necrosis factor a antibodies, mammalian target of rapamycin (mTOR) inhibitors, mycophenolate mofetil, interleukin-2 receptor antibodies. (4) The following agents are suggested as third line treatment options in acute steroid-refractory GVHD: alemtuzumab pentostatin, mesenchymal stem cells and methotrexate. In one aspect, disclosed herein are methods of treating, inhibiting, or preventing GVHD in a subject wherein the subject has acute steroid-refractory GVHD. In such methods, the method can further comprise administering to the subject extracorporeal photopheresis, anti- tumour necrosis factor a antibodies, mammalian target of rapamycin (mTOR) inhibitors, mycophenolate mofetil, interleukin-2 receptor antibodies, alemtuzumab pentostatin,

mesenchymal stem cells, methotrexate, or any combination thereof.

55. Complete responses occur in 25% - 40% of patients treated for acute GVHD, and clinically relevant improvement, defined as regression of skin rash or decrease in the volume of diarrhea and the extent of liver function abnormalities in 40% to 50% of patients with grades II - IV acute GVHD. However, the likelihood of response decreases with increasing severity of the disease.

56. Current pharmacologic and other strategies to prevent GVHD are clearly insufficient. The standard initial treatment for acute GVHD includes injections of glucocorticoids as an immune-suppressive agent. However, the likelihood of this treatment decreases with GVHD severity. Treatment with glucocorticoids, especially long-term treatment, results in impairment of healthy anabolic physiological processes. Resulting adverse side effects include

hyperglycemia, osteoporosis, weight gain, negative calcium balance, and more. The addition of additional immunosupressive drugs to the glucocorticoid regimen has not been shown to be effective.

57. When GVHD progresses in spite of glucocorticoid treatment, or when the treatment cannot be tapered down, secondary treatment may be initiated. However, no single secondary treatment has been shown to be effective, and trial-and-error on an individual basis is the norm. All the secondary treatments have a limited effect and/or result in deleterious side effects. Thus, these therapies are not first line, and most are in clinical trial stage. These secondary treatments include but are not limited to extracorporeal photopheresis, monoclonal antibodies (e.g.

Rituximab), tyrosine kinase inhibitors (e.g. Imatinib), chemotherapeutic drugs (e.g. Pentostatin), and T and B cell inhibitors (e.g. Mycofenolate).

58. In one embodiment, disclosed herein are methods of treating, inhibiting, or preventing GVHD, wherein the subject has grade I acute GVHD under the Glucksberg grading system. Where the subject has grade I acute GVHD, disclosed are methods of treating, inhibiting, or preventing GVHD in a subject comprising administering to the subject adherent stromal cells, compositions comprising adherent stromal cells, and/or medicaments

manufactured using adherent stromal cells further comprising administering to the subject one or more of a topical therapy for skin rash and a calcineurin inhibitor.

59. In another embodiment , disclosed herein are methods of treating, inhibiting, or preventing GVHD, wherein the subject has grade II- IV acute GVHD under the Glucksberg grading system. Where the subject has grade II- IV acute GVHD, disclosed are methods of treating, inhibiting, or preventing GVHD in a subject comprising administering to the subject adherent stromal cells, compositions comprising adherent stromal cells, and/or medicaments manufactured using adherent stromal cells further comprising administering to the subject a corticosteroid.

60. In another embodiment, disclosed herein are methods of treating, inhibiting, or preventing GVHD, wherein the subject has chronic GVHD. Where the subject has chronic GVHD, disclosed are methods of treating, inhibiting, or preventing GVHD in a subject comprising administering to the subject adherent stromal cells (including, for example, PLX cells), compositions comprising adherent stromal cells, and/or medicaments manufactured using adherent stromal cells further comprising administering to the subject an immunosuppressive treatment, such as, for example, methotrexate, cyclosporine, a corticosteroid, and anti-thymocyte globulin.

C. Compositions

61. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular adherent stromal cell is disclosed and discussed and a number of modifications that can be made to the adherent stromal cell, specifically contemplated is each and every

combination and permutation of the composition comprising the adherent stromal cell and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

62. Mesenchymal stem cells (MSCs) possess immunomodulatory and anti-inflammatory properties that may make them an effective therapy for acute and possibly also chronic GVHD. However, several issues limit the use of MSCs. Challenges include a requirement for HLA matching in some cases, the difficulty in obtaining bone-marrow-derived MSCs, and the need to culture the cells to obtain higher cell numbers.

63. Three-dimensionally-adherent cells from bone marrow, placenta, and adipose tissues (See, e.g., International Patent Publication Numbers WO 2007/108003 and WO 2009/037690 [3D -adherent cells by Plurix; PLX cells]) have immunomodulatory and anti-inflammatory capabilities mediated by the secretion of a number of cytokines and chemokines. Thus, PLX cells have the potential of ameliorating or preventing GVHD. PLX cells have a limited differentiation capacity and therefore are not MSCs, which is preferred for GVHD. Moreover, PLX cells do not require HLA matching, are derived from an abundant source, and can be cultured in large numbers and at a low cost. In one aspect, Accordingly, disclosed herein are methods of treating, inhibiting, or preventing GVHD in a subject comprising administering to the subject adherent stromal cells, a medicament manufactured using said adherent stromal cells, and/or compositions comprising said adherent stromal cells wherein the recipient is an HLA- matched related recipient; an HLA-matched unrelated recipient, or an HLA-mismatched recipient. In other words, the source of the adherent stromal cells are an HLA matched related donor, HLA matched unrelated donor, or an HLA-mismatched donor, respectively. Similarly, it is contemplated herein that tissue being grafted in a recipient in the disclosed methods of treating, inhibiting, or preventing GVHD may be from an autologous, syngeneic, allogeneic, or non-autologous source.

64. In one aspect disclosed herein are adherent stromal cells for the treatment, inhibition, or prevention of GVHD or for use in the manufacture of a medicament for the treatment, inhibition, or prevention of GVHD, as well as, compositions comprising said cells. It is understood and herein contemplated that the disclosed adherent stromal cells can be obtained from any source including but not limited to adipose tissue, placental tissue, and bone marrow. 65. It is further contemplated herein that the adherent stromal cells for use in

manufacturing a medicament, as part of a composition, and/or in the method of treating, inhibiting, or preventing GVHD disclosed herein can be propagated using two dimensional ("2D") or three dimensional ("3D") culturing conditions. Nonlimiting examples of such culture conditions are provided in the Detailed Description and in the Examples. Thus, in one aspect, disclosed herein are adherent stromal cells, composition s comprising said cells, uses in manufacturing a medicament, and/or methods of treating, inhibiting, or preventing GVHD wherein the adherent stromal cells (e.g., placental or adipose stromal cells) are cultured under 2D culturing conditions. Similarly, disclosed herein are adherent stromal cells, composition s comprising said cells, uses in manufacturing a medicament, and/or methods of treating, inhibiting, or preventing GVHD wherein the adherent stromal cells (e.g., placental or adipose stromal cells) are cultured under 3D culturing conditions (including, example in a 3D bioreactor).

66. "Two-dimensional" or "2D" refers to a culture in which the cells are grown on a flat tissue culture plate surface (e.g. "TCPS").

67. As used herein the phrase "three dimensional" or "3D" is defined as culture on any surface that has a third dimensionality component. 3D surfaces include but are not limited to porous materials, woven fibers, non-woven fibers, hollow fibers, surfaces with nano or micron scale roughness, sponges, and microcarriers. Other examples of 3D surfaces are given in the examples. It will be appreciated that the conditions of the three-dimensional culture are such that they enable expansion of the adherent cells.

68. As used herein, "ASC-2D" means a culture of adherent stromal cells from any tissue source that have been grown in 2D culture conditions without a period of 3D culture.

69. As used herein, "ASC-3D" means a culture of adherent stromal cells from any tissue source that have been grown in 3D-culture conditions. This term encompasses cells that are grown initially in 2D culture then moved to a 3D culture.

70. "Placental ASC-2D" as used herein is a general term for any culture of placental- derived adherent stromal cells produced using a 2D culture system.

71. "Placental ASC-3D" as used herein is a general term for any culture of placental- derived adherent stromal cells produced using a 3D culture system.

72. As used herein the terms "expanding" and "expansion" refer to an increase of a cell population (e.g. , at least 2 fold), optionally without differentiation accompanying such increase.

73. The cells may be of autologous or non-autologous source (i.e., allogeneic or xenogeneic) of fresh or frozen (e.g., cryo-preserved) preparations. 74. Depending on the medical condition, the subject may be administered with additional chemical drugs (e.g., immunomodulatory, chemotherapy etc.) or cells.

75. In one aspect, it is contemplated herein that the donor source adherent stromal cells does not have to be HLA-matched with the recipient. Accordingly, disclosed herein are adherent stromal cells, medicaments manufactured using said cells, and compositions comprising said cells wherein the recipient is an HLA-matched related recipient; an HLA- matched unrelated recipient, or an HLA-mismatched recipient. In other words, the source of the adherent stromal cells are an HLA matched related donor, HLA matched unrelated donor, or an HLA-mismatched donor, respectively. Similarly, it is contemplated herein that tissue being grafted in a recipient may be from a non-autologous source. Since non- autologous cells may induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immunoisolating, semipermeable membranes before transplantation.

76. Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).

77. Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha- phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

78. For example, microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEM A), methacrylic acid (MA A) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μιη. Such microcapsules can be further encapsulated with additional 2-5 μιη ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S.M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).

79. Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Technol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

80. It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μιη

(Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002;13:783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T.A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

81. In any of the methods described herein, the cells can be administered either per se or, preferably as a part of a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier. Also, as noted above, the cells can be placental-derived, thus, the

compositions and pharmaceutical compositions for use in any of the various methods can be placental-derived adherent stromal cells. The placental-derived ASC may be ASC-3D or ASC- 2D.

82. It is contemplated herein that the adherent stromal cells disclosed in this paper have many utilities associated with the treatment, inhibition, or prevention of GVHD, including, but not limited to a direct use in treatment, inhibition, or prevention of GVHD or in the preparation or manufacture of a medicament for treating, inhibiting, or preventing GVD. Accordingly, in one aspect, disclosed herein is the use of adherent stromal cells for the manufacture of a medicament for treating or preventing GVHD in a subject. It is understood that such adherent stromal cells can be obtained from placental or adipose tissue.

1. Manufacture of 2D Adherent Stromal Cells

83. When ASC-2D cells are used herein, they may be produced by any process using a 2D culture system, such as flasks or plates. In general, cells are seeded into a 2D vessel and allowed to adhere. The first passage is usually carried out after 10-15 days. Beginning at passage 2 and continuing until passage 6-8, cells are passaged when the culture reached 70-80 % confluence, usually after about 3-5 days (1.5-2 doublings). The cells are detached from plates or flasks using 0.25 % trypsin-EDTA (4 minutes at 37 °C) and seeded in a culture density of about 3 + 0.2 x 10³ cells/cm². The size of the tissue culture flasks or plates can be increased as the passaging proceeds. For example, the culturing process may start in an 80 cm² tissue culture flask, continue in 175 cm², and then in 500 cm² (Triple flask). In some embodiments, cells may be re-seeded into Cell Factory 10 tray (6320 cm2).

84. ASC-2D are generally detached from the culture surface with Trypsin-EDTA

(Biological Industries, Beit Ha'emek, Israel; 3-15 minutes with gentle agitation, 1-5 times), and are thereafter resuspended in DMEM and either used directly for testing or other uses or cryopreserved for later testing or use.

85. In one embodiment, the ASC-2D are placental-derived ASC-2D.

2. Manufacture of 3D Adherent Stromal Cells

86. In one aspect, disclosed herein are adherent stromal cells, medicaments manufactured from said adherent stromal cells, and/or compositions comprising adherent stromal cell wherein the adherent stromal cells are derived from placental MSC. In another aspect, disclosed herein are adherent stromal cells, compositions comprising said adherent stromal cells, and/or medicament manufactured with said adherent stromal cells wherein the adherent stromal cells are obtained from a three-dimensional culture (3D) such as a culture in a 3D bioreactor, such as, for example, PluriX™ Plug Flow bioreactor (Pluristem, Haifa, Israel); Celligen™ Plug Flow bioreactor; or Packed Bed Spinner Vessel. Said culturing methods can be effected under perfusion. It is further contemplated herein that the adherent cells can be cultured for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, 1, 2, 3, 4, 5, 6, 7 days, 2, 3, 4, weeks, 2 or 3 months.

87. The skilled artisan can appreciate that the disclosed cultures can occur in any media appropriate for propagating the particular stromal cell population. Non-limiting examples of base media useful in culturing cells to derive ASCs include Minimum Essential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free), FIO(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification), Basal Medium Eagle (BME-with the addition of Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM- without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E-with Earle's sale base), Medium Ml 99 (M199H-with Hank's salt base), Minimum Essential Medium Eagle (MEM-E-with Earle's salt base), Minimum Essential Medium Eagle (MEM-H-with Hank's salt base) and Minimum Essential Medium Eagle (MEM-NAA with non-essential amino acids), among numerous others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. In some embodiments the medium is DMEM. These and other useful media are available from GIBCO, Grand Island, N.Y., USA and Biological

Industries, Bet HaEmek, Israel, among others.

88. The medium may be supplemented such as with serum such as fetal serum of bovine or other species, and optionally or alternatively, growth factors, vitamins (e.g. ascorbic acid), cytokines, salts (e.g. B-glycerophosphate), steroids (e.g. dexamethasone) and hormones e.g., growth hormone, erythropoeitin, thrombopoietin, interleukin 3, interleukin 6, interleukin 7, macrophage colony stimulating factor, c-kit ligand/stem cell factor, osteoprotegerin ligand, insulin, insulin like growth factors, epidermal growth factor, fibroblast growth factor, nerve growth factor, cilary neurotrophic factor, platelet derived growth factor, and bone

morphogenetic protein at concentrations of between picogram/ml to milligram/ml levels.

89. The skilled artisan will appreciate that additional components can be added to the culture medium. Such components may be antibiotics, antimycotics, albumin, amino acids, and other components known to the art for the culture of cells.

90. Examples of adherent materials that may be used to culture cells as described herein include, but are not limited to, a polyester, a polypropylene, a polyalkylene, a

polyfluorochloroethylene, a polyvinyl chloride, a polystyrene, a polysulfone, a cellulose acetate, a glass fiber, a ceramic particle, a matrigel, an extra cellular matrix component (e.g., fibronectin, chondronectin, laminin), a collagen, a hydro gel, a poly L lactic acid and an inert metal fiber. These materials are exemplary only as the material used for the 2D or 3D substratum surface is immaterial so long as it permits the cells to adhere.

91. Even though exemplary culture methods are described for 2D and 3D surfaces, it is the dimensionality of the culture system (3D versus 2D) that is used in the description of the several embodiments that is the relevant factor. Thus, any of a variety of culture methods, including but not limited to suspension bioreactors, packed bed bioreactors, fixed bed bioreactors, rolling flasks, and any method of culturing cells in a liquid environment, may be used in the various aspects and embodiments of the disclosure.

92. Adherent material for the 3D aspect of the present disclosure is configured for 3D culturing thereby providing a growth matrix that substantially increases the available attachment surface for the adherence of the stromal cells so as to mimic the infrastructure of the tissue (e.g., placenta).

93. For example, for a growth matrix of 0.5 mm in height, the increase is by a factor of at least from 5 to 30 times, calculated by projection onto a base of the growth matrix. Such an increase by a factor of about 5 to 30 times, is per unit layer, and if a plurality of such layers, either stacked or separated by spacers or the like, is used, the factor of 5 to 30 times applies per each such structure. When the matrix is used in sheet form, preferably non-woven fiber sheets, or sheets of open-pore foamed polymers, the preferred thickness of the sheet is about 50 to 1000 μιη or more, there being provided adequate porosity for cell entrance, entrance of nutrients and for removal of waste products from the sheet. According to a one embodiment the pores have an effective diameter of 10 μιη to 300 μιη. Such sheets can be prepared from fibers of various thicknesses, in one embodiment the fiber thickness or fiber diameter range is from about 0.5 μιη to 100 μιη, in other embodiments it is in the range of 10 μιη to 15 μιη in diameter.

94. The structures of the 3D culture system may be supported by, or even better bonded to, a porous support sheet or screen providing for dimensional stability and physical strength. Such matrix sheets may also be cut, punched, or shredded to provide particles with projected area of the order of about 0.2 mm² to about 10 mm², with the same order of thickness (about 50 to 1000 μιη). Further details relating to the fabrication, use and/or advantages of the growth matrix which was used to reduce the present disclosure to practice are described in U.S. Pat. No. 5,168,085, and in particular, U.S. Pat. No. 5,266,476, both of which are incorporated herein by reference.

95. The adherent surface which comprises the 3D structure may be of any shape, including but not limited to squares, triangles, rings, disks, balls, ovals, cruciforms and any other shape that can be formed by a flexible or inflexible 3D structure. For high scale production, culturing is preferably effected in a 3D bioreactor.

96. Examples of such bioreactors include, but are not limited to, a plug flow bioreactor, a continuous stirred tank bioreactor and a stationary-bed bioreactor. An example of a three dimensional (3D) plug flow bioreactor is described in U.S. Pat. No. 6,911,201 that is capable of supporting the growth and prolonged maintenance of stromal cells. In this bioreactor, stromal cells are seeded on porous carriers made of a non-woven fabric matrix of polyester, packed in a glass column, thereby enabling the propagation of large cell numbers in a relatively small volume.

97. The matrix used in the plug flow bioreactor can include, but is not limited to, sheet form, non-woven fiber sheets, or sheets of open-pore foamed polymers, the preferred thickness of the sheet is about 50 to 1000 μιη or more, there being provided adequate porosity for cell entrance, entrance of nutrients and for removal of waste products from the sheet.

98. Other examples of 3D bioreactors include, but are not limited to, a plug flow bioreactor, a continuous stirred tank bioreactor, a stationary-bed bioreactor, a CELLIGEN PLUS® bioreactor system (New Brunswick Scientific (NBS), and a BIOFLO 310 bioreactor system (New Brunswick Scientific (NBS). Other examples of bioreactors include an air-lift bioreactor where air is typically fed into the bottom of a central draught tube flowing up while forming bubbles; a cell seeding perfusion bioreactor with Polyactive foams [as described in Wendt, D. et al., Biotechnol Bioeng 84: 205-214, (2003)], tubular poly-L-lactic acid (PLLA) porous scaffolds in a radial-flow perfusion bioreactor [as described in Kitagawa et al.,

Biotechnology and Bioengineering 93(5): 947-954 (2006). Still other bioreactors which can be used are described in U.S. Pat. Nos. 6,277,151, 6,197,575, 6,139,578, 6,132,463, 5,902,741 and 5,629,186.

99. In general, bioreactors are capable of 3D expansion of adherent cells under controlled conditions (e.g., pH, temperature and oxygen levels) and with constant cell growth medium perfusion. Furthermore, the cell cultures can be directly monitored for concentration levels of glucose, lactate, glutamine, glutamate and ammonium. The glucose consumption rate and the lactate formation rate of the adherent cells enable to measure cell growth rate and to determine the harvest time.

100. Cell seeding is preferably effected at a concentration of 20,000-1,500,000 cells / ml at seeding. In an exemplary embodiment a total of 150 + 30 x 10⁶ cells are seeded, 3-5 x 10⁶ cell/g carrier are seeded, or 0.015-0.1 x 10⁶ cell/ml are seeded.

101. Cells can be harvested when at least about 10% of cells are proliferating while avoiding uncontrolled differentiation and senescence. In one aspect disclosed herein are methods of treating, inhibiting, and or preventing GVHD wherein the adherent stromal cells, compositions comprising said cells, and/or medicaments manufactured using said cells wherein culturing of the adherent cells is effected until at least about 10% of the adherent cells are proliferating.

102. Culturing of adherent cells in the disclosed methods of treating, inhibiting, or preventing GVHD or uses for manufacture of a medicament can be effected for at least about 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, a month or even more. It will be appreciated that culturing in a bioreactor can prolong this period.

103. Adherent cells of some embodiments of the present disclosure comprise at least about 10%, 28%, 30%, 50%, 80% or more proliferative cells (as can be assayed by FACS monitoring S and G2/M phases).

104. In order to provide 3D and 2D adherent cells, various manufacturing systems may be used. The examples provided below are illustrative only and additional methods are provided in the Examples. In any of the culture systems described that utilize placenta as the cells source, all placentas obtained were received from the maternity ward under approval of the Helsinki Committee of the medical facility. Accordingly, all placenta donors signed an informed consent and Donor Screening and Donor Testing was performed.

105. In general, to initiate any of the culture processes that involve placenta, the whole placenta is cut into pieces under aseptic conditions under laminar flow hood, washed with Hank's buffer solution and incubated for 3 hours at 37 °C with 0.1 % Collagenase (1 mg

Collagenase/ml tissue). 2D cell medium (2D-Medium comprising DMEM supplemented with 10 % FBS, fungizone 0.25 μg/ml and gentamycine 50 μg/ml) is added and the digested tissue is roughly filtered through a sterile metal strainer, collected in a sterile beaker and centrifuged (10 minutes, 1200 RPM, 4 °C). Using gentle pipeting, suspended cells are then washed with 2D- Medium supplemented with antibiotics, seeded in 80 cm2 flasks and incubated at 37°C in a tissue culture incubator under humidified condition supplemented with 5 % C02. Following 2-3 days, in which the cells were allowed to adhere to the flask surface, they are washed with PBS and 2D-Medium was added.

106. While placenta is one source of ASC and is the ASC source used in the

Examples, placenta is an exemplary source and other cells sources may be used. Examples of other ASC sources include adipose tissue, umbilical cord, blood, and bone marrow. Thus, in the various aspects and embodiments of the disclosure the ASC are not limited to placenta-derived ASC. However, in one embodiment, the ASC-2D, the ASC-3D, or both the ASC-2D and ASC- 3D are placenta-derived ASC.

a) Manufacture of 3D Adherent Cells by PluriX™

107. In one embodiment, the ASC-3D cells are produced using a PluriX™ Plug Flow bioreactor (Pluristem, Haifa, Israel) as illustrated in U.S. Pat. No. 6,911,201. In general, the PluriX™ Plug Flow bioreactor is loaded with 1-100 ml packed 3D porous carriers (4 mm in diameter) made of a non- woven fabric matrix of polyester. These carriers enable the

propagation of large cell numbers in a relatively small volume. The bioreactor is maintained in an incubator of 37°C, with flow rate regulated and monitored by a valve, and peristaltic pump. The 02 proportion is suited to the level of dissolved 02 at the bioreactor exit, determined by a monitor.

108. Non-confluent primary human adherent 2D cell cultures are trypsinized, washed, resuspended in DMEM supplemented with 10% FBS, Pen-Strep-Nystatin mixture and 2 mM L- glutamine, and seeded (10³-10⁵ cells/ml) via an injection point onto the 3D carriers in a sterile Plug Flow bioreactor. Prior to inoculation, the bioreactor is generally filled with PBS-Ca— Mg (Biological Industries, Beit Ha'emek, Israel), autoclaved (120°C, 30 min) and washed with Dulbecco's growth medium containing 10% heat-inactivated fetal calf serum and a Pen-Strep- Nystatin mixture. Flow is kept at a rate of about 0.1-5 ml/min. The seeding process generally involves cessation of circulation for 2-48 hrs, which allow the cells to settle on the carriers. The bioreactor is generally kept under controlled temperature (37°C) and pH conditions (pH=6.7- 7.4); using an incubator supplied with sterile air and C02 as needed. Growth medium is replaced 2-3 times a week. Circulation medium is replaced with fresh DMEM media, every 4 hr to 7 days. At a density of about Ixl0⁶-lxl0⁷ cells/ml (generally following 12-40 days of growth), the total medium volume is removed from the bioreactor and bioreactor and carriers are washed 3-5 times with PBS. PluriX™ 3D-adherent cells are then detached from the carriers with Trypsin-EDTA; (Biological Industries, Beit Ha'emek, Israel; 3-15 minutes with gentle agitation, 1-5 times), and are thereafter resuspended in DMEM and either used directly for testing or other uses or cryopreserved for later testing or use.

109. In one embodiment, the PluriX™ ASC-3D are placental-derived ASC-3D.

b) Manufacture of 3D Adherent Cells by Celligen™

110. In another embodiment, the ASC-3D cells are produced using a Celligen™ Plug Flow bioreactor, as illustrated in US 2010/0209403 and WO 2009/037690. Generally speaking, the 3D growth phase is performed using an automatic CelliGen Plus® or BIOFLO 310 bioreactor system [(New Brunswick Scientific (NBS)] depicted in Figure 8C of US

2010/0209403. The parameters of the process are monitored and controlled by a control console which included connectors for probes, motor and pumps, control loops for Dissolved Oxygen (DO), pH, perfusion and agitation (with a motor), a gases control system, water circulation and heating system for temperature control and an operator interface. The controlled process parameters (such as temperature, pH, DO etc.) can be displayed on the operator interface and monitored by a designated controller.

111. Generally, about 150 + 30 x 106 cells cryopreserved ASC-2D are thawed, washed and seeded in a sterile bioreactor. The bioreactor generally contains 30-50 gr carriers

(FIBRACEL® disks, NBS), made of Polyester and Polypropylene and 1.5 + 0.1 L 3D-Medium. The growth medium in the bioreactor is kept at the following conditions: 37 °C, 70 % Dissolved Oxygen (DO) and pH 7.3. Filtered gases (Air, C02, N2 and 02) are supplied as determined by the control system in order to keep the DO value at 70 % and the pH value at 7.3. For the first 24 hours, the medium is usually agitated at 50 Rounds Per Minute (RPM) and increased up to 200 RPM by day 2. For the first 2-3 days, the cells are grown in a batch mode. Perfusion is initiated when the medium glucose concentration decreases below 550 mg/liter. The perfusion is adjusted in order to keep the glucose concentration constant at approximately 550 + 50 mgUiter. The glucose consumption rate and the lactate formation rate of the cell culture enables measure of the cell growth rate. These parameters are used to determine the harvest time based on accumulated experimental data.

112. The cell harvest process starts at the end of the growth phase (usually 4-10 days). The 3D-grown culture is usually harvested by emptying the bioreactor vessel using gravitation via tubing to a waste container. The vessel is opened and the carriers aseptically transferred from the basket to the upper basket net. The bioreactor vessel is then closed and refilled with pre-warmed PBS (37°C). The agitation speed is increased to about 150 RPM for 2 minutes. The PBS is then drained and this washing procedure repeated twice.

113. In order to release the cells from the carriers, generally 1.5 L pre-warmed to 37 °C Trypsin-EDTA (Trypsin 0.25 %, EDTA 1 mM) is added to the bioreactor vessel and carriers are agitated for 5 minutes in 150 RPM, 37 °C. The cell suspension is collected to a sterile container containing 250 ml FBS. The cell suspension ("PLX-C") is then divided or further processed as needed for testing and use.

114. In one embodiment, the Celligen™ ASC-3D are placental-derived ASC-3D.

c) Manufacture of 3D Adherent Cells by Packed Bed Spinner Vessel

115. In one embodiment, the ASC-3D cells are produced using a packed bed spinner flask. The packed is based on a 500ml glass spinner flask with a magnetic stirrer. The spinner flask if fitted with a packed bed apparatus similar to the Celligen™ Plug Flow bioreactor (see above) which is packed with 1.8gr of FIBRACEL (or other carriers). The spinner is batch fed (rather than by perfusion), fitted with two 0.22μιη filters, and placed in a 37°c 5% C02 incubator. Cells are seeded onto the scaffold by introducing to the medium and allowing 4 hours of 40 RPM agitation. Subsequently the RPM is increased to 120 RPM. Medium is assessed daily for glucose level and replaced to maintain acceptable glucose concentration. At the end of the culture process, carriers are removed from the packed bed, washed twice with PBS, and processed or removed from the carriers by agitation and enzymatic digestion for further use.

3. Pharmaceutical carriers/Delivery of pharmaceutical products

116. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. 117. The adherent stromal cells, medicaments manufactured using said adherent stromal cells, and/or compositions comprising said adherent stromal cells may be administered systemically or locally (including, for example, at the site of tissue transplantation or site of GVHD damage) in the disclosed methods of treating, inhibiting, and/or preventing GVHD disclosed herein. The adherent stromal cells, medicaments, and/or compositions can be administered by any route suitable for the delivery of adherent stromal cells, medicaments, and/or composition comprising adherent stromal cells in the disclosed methods of treating, inhibiting, or preventing GVHD including, but not limited to orally, sublingually, rectally, parentally (e.g., intravenous injection (i.v.), intracranial injection (i.e.); intramuscular injection (i.m.), intraperitoneal injection (i.p.), and subcutaneous injection (s.c.) and intraosseous infusion (i.o.)), transdermally, extracorporeally, inhalation, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the composition through use of a nebulizer or like device.

Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. Oral delivery can be through pill, tablet, liquid, or sublingual droplets. Topical administration can be through ointment, gel, hydrogel, paste, film, cream, lotion, transdermal patch, dermal patch, shampoo, or liposomes. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

118. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

119. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.

Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

120. The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier or excipient.

121. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

122. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

123. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.

Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

124. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously,

intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

125. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

126. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

127. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.. 128. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

b) Therapeutic Uses

129. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the adherent stromal cells used alone might range from about 10 million to about 500 million cells per administration, depending on the factors mentioned above. For example, the dosage of adherent stromal cells can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 million cells or any amount in between. It is further understood that a range of adherent stromal cells can be used including from about 10 to about 500 million cells, from about 100 to about 400 million cells, from about 150 to about 300 million cells. Accordingly, disclosed herein are methods of treating, inhibiting, or preventing GVHD in a subject in need thereof, the method comprising administering to said subject a therapeutically or prophylactically effective amount of adherent stromal cells, wherein the dosage of adherent stromal cells administered to the subject is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 million cells or from about 150 million to about 300 million cells. Adherent stromal cells, compositions comprising adherent stromal cells, and/or medicaments

manufactured using said adherent stromal cells can be administered for a duration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, 5 years, or more.

130. It is contemplated herein that it can be advantageous to administer more than a single dose of the adherent stromal cells, compositions comprising said adherent stromal cells, or medicaments manufactured using said adherent stromal cells. In one aspect, the disclosed cells and compositions can be administered singly or in multiple administrations. Thus, disclosed herein are methods of treating GVHD and uses comprising administering the disclosed adherent stromal cells, medicaments, and/or compositions comprising said cells wherein the adherent stromal cells, compositions comprising said adherent stromal cells, or medicament manufactured using said adherent stromal cells are administered 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 24, 25, 30, 35, 36, 40, 45, 48, 50, 55, 60 or more times. When more than a single administration, the adherent stromal cells or compositions comprising said cells may be administered at regular or variable intervals. For example, the adherent stromal cells or compositions comprising said cells may be administered regularly such that they or administered hourly, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours; daily, every other day; every third day, weekly, every two weeks, monthly, or yearly. Alternatively, cells or compositions comprising said cells can be administered according to the methods at irregular (i.e., variable intervals) such as, for example, a first dose, a second dose a week later, and a third dose a month following the first dose later; or a first dose followed by a second dose a month later and a third dose three months following the first dose; or doses at one week intervals for three months, followed by monthly intervals until the one year point, followed by yearly doses.

131. Additionally, it is contemplated herein that the disclosed compositions can be administered before, after, concurrently with, or in combination with a graft when used in the disclosed methods and uses for treating GVHD. For example, the disclosed adherent stromal cells, compositions comprising said adherent stromal cells, or medicament manufactured using said adherent stromal cells can be administered 7, 6, 5, 4, 3, 2, days, 24, 23, 22, 21, 20, 19, , 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours, 45, 30, 15, 10, 5, 4, 3, 2, or 1 min or any combination thereof before administration of the graft in the disclosed methods and uses of treating GVHD. Alternatively, the disclosed adherent stromal cells, compositions comprising said adherent stromal cells, or medicament manufactured using said adherent stromal cells can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60min, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 20, 24, 25, 30, 36, 40, 45, 48, 50, 55, or 60 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, or 60 months or any combination thereof after administration of the graft or onset of GVHD in the disclosed methods and uses of treating GVHD. It is further understood that adherent stromal cells, compositions comprising said adherent stromal cells, or medicament manufactured using said adherent stromal cells can be administered in combination before, concurrently with, and/or after administration of the graft in the disclosed methods and uses of treating GVHD.

132. It is understood and herein contemplated that additional agents may need to be administered along with the adherent stromal cells to facilitate engraftment or further guard against GVHD. Accordingly, disclosed herein are adherent stromal cells or composition comprising adherent stromal cells administered in the methods or uses disclosed herein wherein the adherent stromal cells administered in a composition for treating, inhibiting, preventing GVHD further comprising an immunosuppressive agent, steroid, anti-inflammatory, small molecule, pharmaceutical, or other agent. For example, the adherent stromal cells or

compositions comprising said cells can be in a further composition comprising topical therapy for skin rash; a calcineurin inhibitor; a corticosteroid; immunosuppressive treatment (e.g., methotrexate, cyclosporine, a corticosteroid, and/or anti-thymocyte globulin); anti-TNF alpha antibody; mammalian target of rapamycin (mTOR) inhibitor; mycophenolate mofetil, interleukin-2 receptor antibody; alemtuzumab pentostatin; and/or mesenchymal stem cells.

133. Following administration of a disclosed composition, such as adherent stromal cells (e.g., PLX cells), for treating, inhibiting, or preventing GVHD, the efficacy of the therapeutic composition comprising adherent stromal cells (e.g., PLX cells) can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a composition, such as a therapeutic composition comprising adherent stromal cells (e.g., PLX cells), disclosed herein is efficacious in treating, preventing, or inhibiting GVHD in a subject by observing that the composition reduces rashes, blistering, nausea, loss of apetite, dryness, jaundice, hair loss, scarring of lung, liver, or intestinal tract, diminished bile flow, or other symptoms of GVHD in the subject.

4. Kits

134. Disclosed herein are kits and articles of manufacture that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits and articles of manufacture can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. The kits and articles of manufacture may further comprise adherent stromal cells. In another aspect, the kits and articles of manufacture may comprise a label, instructions, and packaging material. For example, disclosed is a kit for or an article of manufacture comprising a packaging material which comprises a label for use in treating or preventing GVHD, said packaging material packaging a pharmaceutically effective amount of adherent stromal cells, wherein the cells are optionally obtained from placenta or adipose tissue. Also disclosed are kits or articles of manufacture which provide a means for extracting adherent cells from a donor source. In another aspect the kits or articles of manufacture can comprise 2D and/or 3D culturing instructions, growth media, and/or containers (such as a 3D bioreactor) for the propagation of adherent stromal cells.

135. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

D. Examples

136. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. Example 1

137. A review by Castor et al. (2012) lists and describes GVHD mouse models.

Furthermore, specific models exist for acute and chronic GVHD. For aGVHD, models include MHC-mismatched models, MiHA-mismatched models, xenogenic transplant models, and antigen-specific transgenic models. For chronic GVHD (cGVHD), models include

sclerodermatus models, autoantibody-mediated models, and a defective thymic function model.

GVHD animal models

138. Animal models are useful for performing mechanistic, kinetic, and therapeutic studies related to GVHD. Most knowledge of the role of the immune system GVHD comes from experiments in mice. Most animal GVHD models utilize whole body irradiation, otherwise known as myeloablative conditioning. Following lethal irradiation, animals are infused with T-cell-depleted bone marrow supplemented with donor lymphocytes (either splenocytes or lymph node T cells). The bone marrow enables hematopoietic reconstitution after transplant. T-cells are depleted from the bone marrow to control the number of immune cells that are delivered.

139. The severity of the GVHD in the model depends on several factors: (1) dose, source and type of T-cell subsets that are delivered with donor bone marrow; (2) irradiation dose; (3) mouse strain (inbred strains are more susceptible to radiation); (4) genetic disparities. For example, even mice that are major histocompatibility complex (MHC)-matched can vary by minor histocompatibility antigens (MiHAs) that influence aGVHD severity; (5) variation in environmental pathogens between labs.

140. Two notable models of GVHD are NOD-scid IL-2Rynull mice and C57BL to

C57BL/6xBALB/c Fl mice, described below.

141. NOD-scid IL-2Rynull mice are immunodeficient due to a mutation in the IL-2 receptor. Thus, they engraft xenogeneic cells readily and can serve as a human-into-mouse xenogeneic GVHD model in which the engrafted cells are amenable to regulation by various therapies, including cell therapies. Development of GVHD in this model is based on cell reactivity with foreign MHC class I and class II. This resembles HLA-mismatched

hematopoietic stem cell transplantation (HSCT) in clinical GVHD where donor cell

alloreactivity is triggered by recognition of foreign MHC antigens expressed by recipient cells.

142. C57BL to C57BL/6xBALB/c Fl mice serve as a semi-allogeneic BM

transplantation model. This model mimics a haplo-identical transplantation (parent-to-child). To induce acute GVHD, spleen cells from C57BL/6 donor mice are injected intravenously into irradiated C57BL/6 Balb/c Fl recipient mice. GVHD develops two to three weeks posttransplantation and includes weight loss, hair loss, and sometimes diarrhea.

143. The ability of adherent stromal cells to prevent GVHD resulting from bone marrow transplantation was tested using C57BL to C57BL/6xBALB/c Fl mice, a semi- allogeneic bone marrow transplantation model. This model mimics transplantation from haplo- identical donor (parent-to-child) in humans, which is the last treatment option, as total mismatched donors are not used. GVHD in this model starts to develop two to three weeks posttransplantation and includes weight loss, hair loss and sometimes diarrhea.

144. The following working solutions were used: Table IV: Working Solutions

All of the ab ove solutions were store d in 4-8 C before use.

145. Ten- to eleven-week-old Fl (Balb/c x C57BL/6) female mice, having a weight of 20-23 grams each, were divided into three groups of six as shown in Table V.

Table V: Experimental Groups

General Experimental Design

146. At day 1, the mice received a whole body lethal irradiation (lOOOrad) in a single dose, and one day after (at day 0) were transplanted with 8 x 10⁶ C57BL/6 bone marrow cells and 10 x 10⁶ spleen cells. PLX cells were intramuscularly (IM) or intravenously (IV) injected into the mice at the day of transplantation (day 0), approximately 20 hours after irradiation, and at day 4. Intramuscular administration was performed by injecting 1 x 10⁶ PLX cells in 50 μΐ PlasmaLyte A for each animal or the same volume of vehicle (PlasmaLyte A) for group A control mice.

147. Intravenous administration of PLX cells (1 x 10⁶ cells) at day 0 was performed through the tail vein after mixing 50 μΐ of PLX suspension (1 x 10⁶ cells) together with the BM and spleen cells in PlasmaLyte, reaching a total volume of 250 μΐ. 1 x 10⁶ PLX cells were intravenously injected also at day 4 in a total volume of 250 μΐ PlasmaLyte while the same volume of PlasmaLyte was administered to the control group.

148. The clinical condition of the mice was monitored every other day for up to six weeks after transplantation, and included monitoring of: 1) body weight, 2) fur loss, and 3) diarrhea. GVHD score were calculated based on body weight, the size of the area of fur loss, and extent of diarrhea.

149. Chimerism of the appropriate HLAs in the blood of three representative mice from each group was tested two weeks after transplantation using FACS. Detailed Steps and Conditions

Mice acclimation

150. Mice (10-11 weeks old Fl (Balb/c x C57BL/6) female mice) were purchased from Harlan. Animals were housed for 5 days in a specific pathogen free (SPF) facility for acclimation before treatment.

GVHD induction

151. Fl mice were given lethal whole body irradiation by a single dose of 1000 rad. One day after irradiation, the mice received a transplant of 8 x 10⁶bone marrow cells and 10 x 10⁶ spleen cells from C57BL/6 mice.

PLX thawing and preparation for administration

152. PLX cells were thawed and prepared for administration briefly as follows. Cells were removed from liquid nitrogen storage, thawed at 37 degrees C, and combined with a solution of human albumin in plasmaLyte A (one volume thawed cells combined with 10 volumes of a combination of 1 part human albumin 20% with 3 parts plasmaLyte A). The cells were gently centrifuged, resuspended in cold plasmaLyte A, recentrifuged, and finally resuspended in cold plasmaLyte A to a final cell concentration of 20 x 10⁶ cells/ml.

PLX administration

153. PLX cells were intramuscularly or intravenously administered at days 0 and at day 4 as follows:

154. Intramuscular administration was performed by injecting 50 μΐ of PLX cell suspension (1 x 10⁶ cells) or PlasmaLyte A per animal, immediately after BM and spleen cells transplantation.

155. Intravenous administration was performed at day 0 by injecting 1 x 10⁶ PLX cells in 50 μΐ PlasmaLyte with bone marrow and spleen cells in PlasmaLyte to reach a total volume of 250 μΐ. At day 4, 1 x 10⁶ PLX cells were injected alone in a total volume of 250 μΐ PlasmaLyte. At both injection time points, the cells were administered slowly via the tail vein (over 1-2 minutes), and the injection times were recorded.

156. In control mice, three mice received intramuscular administration with 50 μΐ PlasmaLyte at days 0 (immediately after BM and spleen cell transplantation) and at day 4, while three other control mice received intravenous administration with bone marrow and spleen cells in 250 μΐ PlasmaLyte at day 0 and 250 μΐ PlasmaLyte only at day 4.

157. For all cell injections, cells were mixed gently during injection to prevent aggregation.

158. Cells were gently mixed all along the injection step to prevent aggregation. Mice monitoring

159. Mice were monitored in SPF conditions by monitoring 1) body weight monitoring, 2) fur loss, and 3) diarrhea. Chimerism in the blood of 3 representative mice from each experimental group was also tested two weeks after transplantation using FACS for H-2Dd allele (expressed by balb/c only).

Mice sacrificing

160. Mice were sacrificed at 5-6 weeks following model induction / PLX

administration, depending on mice clinical condition. If the clinical condition of one or more mice deteriorated before the end of the experiment, mice were sacrificed according to the rules of the ethical approval. The parameters monitored were weight (% of initial weight) and GVHD score. Skin, liver, and colon samples were also collected for histology and were preserved in 10% neutral-buffered formalin for optional paraffin embedding.

Results

161. Compared to treatment with vehicle alone, PLX cell treatment improved the mice's recovery from irradiation and GVHD induction by C57BL/6 bone marrow and spleen cell transplantation. Mice administered PLX cells either intramuscularly (IM) or intravenously (IV) recovered body weight more quickly and to a greater extent than control (vehicle-treated) mice (Figure 1), and the PLX mice showed improved clinical conditions. The IV mice improved slightly more quickly than the IM mice. The body weight differences between all groups persisted until approximately four weeks after GVHD induction. The rate of survival of PLX-treated mice was also slightly greater than control mice (Figure 2A). PLX treatment did not alter the rates of HLA chimerism in the mice (Figure 2B).

162. Clinical signs of GVHD appeared in the mice beginning 15 days after transplantation. PLX cells markedly reduced GVHD score in both IM and IV PLX-treated mice groups up to six weeks after transplantation (Figure 3). PLX cells significantly reduced skin

GVHD in the IM mice and virtually eliminated it the IV mice (Figure 4; for median score at day 32 [lower panel], a statistically significant difference was apparent between PLX-treated and control cells). For mice surviving by days 36 and 43, a statistically significant difference in GVHD score was apparent between PLX-treated and untreated groups in GVHD scores (Figure 5; in lower panels, values for IM and IV PLX-treated mice are combined). Photographs of test mice show the fur loss associated with skin GVHD that occurred in several control mice and an IM PLX-treated mouse but not in normal mice or IV PLX-treated mice (Figure 6, arrows show mice with fur loss). Thus, PLX cells prevented several GVHD clinical signs in semi-allogeneic GVHD. E. References

Inamoto, Y. and M.E. Flowers, Treatment of chronic graft-versus-host disease in 2011. Curr Opin Hematol, 2011. 18(6): p. 414-20.

Perez, L., C. Anasetti, and J. Pidala, Have we improved in preventing and treating acute graft-versus- host disease? Curr Opin Hematol, 2011. 18(6): p. 408-13.

Castor, M.G., V. Pinho, and M.M. Teixeira, The role of chemokines in mediating graft versus host disease: opportunities for novel therapeutics. Front Pharmacol, 2012. 3: p. 23.

Przepiorka, D., et al., 1994 Consensus Conference on Acute GvHD Grading. Bone Marrow

Transplant, 1995. 15(6): p. 825-8.

Rowlings, P. A., et al., IBMTR Severity Index for grading acute graft-versus-host disease:

retrospective comparison with Glucksberg grade. Br J Haematol, 1997. 97(4): p. 855-64.

Glucksberg, H., et al., Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation, 1974. 18(4): p. 295-304.

Cahn, J.Y., et al., Prospective evaluation of 2 acute graft-versus-host ( GvHD) grading systems: a joint Societe Francaise de Greffe de Moelle et Therapie Cellulaire (SFGM-TC), Dana Farber Cancer Institute (DFCI), and International Bone Marrow Transplant Registry (IBMTR) prospective study. Blood, 2005. 106(4): p. 1495-500.

Dignan, F.L., et al., Diagnosis and management of acute graft-versus-host disease. Br J Haematol, 2012. 158(1): p. 30-45.

Ali, N., et al., Xenogeneic graft-versus-host-disease in NOD-scid IL-2Rgammanull mice display a T- effector memory phenotype. PLoS One, 2012. 7(8): p. e44219.

Ilan, Y., et al., Alleviation of acute and chronic graft-versus-host disease in a murine model is associated with glucocerebroside-enhanced natural killer T lymphocyte plasticity. Transplantation, 2007. 83(4): p. 458-67.

Claims

V. CLAIMS What is claimed is:

1. A method of treating or preventing graft-versus-host disease (GVHD) in a subject in need thereof comprising administering to said subject a therapeutically or prophylactically effective amount of adherent stromal cells, thereby treating or preventing the GVHD.

2. Use of adherent stromal cells for the manufacture of a medicament for treating or preventing GVHD in a subject, wherein the cells are optionally obtained from placenta or adipose tissue.

3. Adherent stromal cells for the treatment or prevention of GVHD, wherein the cells are optionally obtained from placenta or adipose tissue.

4. An article of manufacture comprising a packaging material which comprises a label for use in treating or preventing GVHD, said packaging material packaging a

pharmaceutically effective amount of adherent stromal cells, wherein the cells are optionally obtained from placenta or adipose tissue.

5. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the subject has acute GVHD.

6 . The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the subject has grade I acute GVHD under the Glucksberg grading system.

7. The method, use, adherent stromal cells, or article of manufacture of claim 6, wherein the subject is receiving one or more of a topical therapy for skin rash and a calcineurin inhibitor.

8. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the subject has grade II- IV acute GVHD under the Glucksberg grading system.

9. The method, use, adherent stromal cells, or article of manufacture of claim 8, wherein the subject is receiving a corticosteroid.

10. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the subject has chronic GVHD.

11. The method, use, adherent stromal cells, or article of manufacture of claim 5 or claim 10, wherein the subject is receiving an immunosuppressive treatment.

12. The method, use, adherent stromal cells, or article of manufacture of claim 11, wherein the subject is receiving one or more of methotrexate, cyclosporine, a corticosteroid, and antithymocyte globulin.

13. The method, use, adherent stromal cells, or article of manufacture of claim 10, wherein the subject requires immunosuppressive treatment for a period of one or more years.

14. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the subject has steroid-refractory GVHD.

15. The method, use, adherent stromal cells, or article of manufacture of claim 14, wherein the subject is receiving one or more of extracorporeal photophoresis, anti-TNF alpha antibody, mammalian target of rapamycin (mTOR) inhibitor, mycophenolate mofetil, interleukin-2 receptor antibody, alemtuzumab pentostatin, mesenchymal stem cells, and methotrexate.

16. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the subject has received or will receive a hematopoietic stem cell transplant.

17. The method, use, adherent stromal cells, or article of manufacture of claim 16, wherein the subject has received or will receive a bone marrow, peripheral blood stem cell, or cord blood transplant.

18. The method, use, adherent stromal cells, or article of manufacture of claim 17, wherein the subject has received or will receive a bone marrow transplant.

19. The method, use, adherent stromal cells, or article of manufacture of claim 18, wherein the subject has received or will receive whole body irradiation.

20. The method, use, adherent stromal cells, or article of manufacture of claim 18, wherein the subject is female.

21. The method, use, adherent stromal cells, or article of manufacture of claim 16, wherein the subject has received or will receive a transplant from an HLA-matched related donor or an HLA-matched unrelated donor.

22. The method, use, adherent stromal cells, or article of manufacture of claim 16, wherein the subject has received or will receive a transplant from an HLA-mismatched related or unrelated donor.

23. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2,

3, or 4, wherein the GVHD is prevented by administering the adherent stromal cells to the subject before the subject receives a tissue transplant.

24. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the GVHD prevented by administering the adherent stromal to the subject at the same time that the subject receives a tissue transplant.

25. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the GVHD is prevented or treated by administering the stromal cells to the subject after the subject has received a tissue transplant.

26. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2,

3, or 4, wherein the adherent cells are administered in a single administration.

27. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the adherent cells are administered in multiple administrations.

28. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the adherent cells are administered at variable intervals.

29. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the adherent cells are administered with a dosage of from about 150 million to about 300 million cells.

30. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the adherent cells are administered systemically.

31. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the adherent cells are administered locally.

32. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the adherent cells are administered locally at the site of a tissue transplant.

33. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2,

3, or 4, wherein the adherent cells are administered by one or more of intramuscular, intravenous, intraperitoneal, subcutaneous, inhalational, or intraosseous infusion routes.

34. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the adherent cells comprise a positive marker expression selected from the group consisting of CD73, CD90, CD29 and CD 105.

35. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2,

3, or 4, wherein the adherent cells comprise a negative marker expression selected from the group consisting of CD3, CD4, CD45, CD80, HLA-DR, CDl lb, CD14, CD19, CD34 and CD79.

36. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2,

3, or 4, wherein the adherent stromal cells are obtained from a three-dimensional (3D) culture.

37. The method, use, adherent stromal cells, or article of manufacture of claim 36, wherein the three-dimensional (3D) culture comprises a 3D bioreactor.

38. The method, use, adherent stromal cells, or article of manufacture of claim 36, wherein culturing of the adherent cells in the 3D culture is effected under perfusion.

39. The method, use, adherent stromal cells, or article of manufacture of claim 36, wherein culturing of the adherent cells is effected for at least 3 days.

40. The method, use, adherent stromal cells, or article of manufacture of claim 36, wherein culturing of the adherent cells is effected until at least 10 % of the adherent cells are proliferating.

41. The method, use, adherent stromal cells, or article of manufacture of claim 36, wherein the adherent cells comprise cells cultured from the placenta or adipose tissue under 2 dimensional (2D) culturing conditions.

42. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the adherent stromal cells are autologous to the subject.

43. The method, use, adherent stromal cells, or article of manufacture of claim 1, 2, 3, or 4, wherein the adherent stromal cells are allogeneic to the subject.